Studies of Murine Olfactory Systems

Most animals rely on the olfactory system to detect chemical information about their environment to properly respond to cues. In mice, two systems, the main olfactory system and the accessory olfactory system, detect odors and relay information about food, danger, predators, and other animals. The main olfactory system consists of olfactory sensory neurons (OSNs) located in the nose, which detect odors that diffuse into the nasal cavity and send signals to the olfactory bulb of the brain. OSNs express one receptor and precisely target to respective regions of the olfactory bulb. These targeting events are finely regulated and the factors that influence these events are of great interest to study. Olfactory ensheathing cells (OECs) surround OSN axons along their targeting routes and have been shown to promote proper axon extension and growth. I have identified the unique expression of a glutamate receptor subunit, GluN3A, in OECs and using genetic methods, ablated GluN3A in these cells and studied the consequences in OSN axon targeting to the olfactory bulb. The accessory olfactory system consists of vomeronasal sensory neurons (VSNs) that are located in a sequestered organ found in the nasal cavity, called the vomeronasal organ (VNO). These neurons detect pheromones from other animals to elicit distinct mating and aggressive behaviors in many species, and project their axons to a specialized region of the olfactory bulb called the accessory olfactory bulb (AOB). This system is less studied than the main olfactory system, and I sought to characterize the process of pheromone delivery to these neurons. Since the VNO is isolated from the environment, the method of pheromone delivery is an active process. I have identified the expression of phosphodiesterase 5A (PDE5A) in the VNO blood vessel, and using pharmacological methods to inhibit PDE5A, showed that the activity of this enzyme is important for pheromone delivery to the VNO. Overall, I have analyzed two proteins that have not been previously characterized in the olfactory system of mice and have shown the consequences of perturbing these proteins in these respective olfactory systems

Lycium barbarum (wolfberry) polysaccharide facilitates ejaculatory behaviour in male rats

Poster Session AOBJECTIVE: Lycium barbarum (wolfberry) is a traditional Chinese medicine, which has been considered to have therapeutic effect on male infertility. However, there is a lack of studies support the claims. We thus investigated the effect of Lycium barbarum polysaccharide (LBP), a major component of wolfberry, on male rat copulatory behavior. METHOD: Sprague-Dawley rats were divided into two groups (n=8 for each group). The first group received oral feeding of LBP at dosage of 1mg/kg daily. The control group received vehicle (0.01M phosphate-buffered saline, served as control) feeding daily for 21 days. Copulatory tests were conducted at 7, 14 and 21 days after initiation of treatment. RESULTS: Compared to control animals, animals fed with 1mg/kg LBP showed improved copulatory behavior in terms of: 1. Higher copulatory efficiency (i.e. higher frequency to show intromission rather than mounting during the test), 2. higher ejaculation frequency and 3. Shorter ejaculation latency. The differences were found at all time points (Analyzed with two-tailed student’s t-test, p<0.05). There is no significant difference found between the two groups in terms of mount/intromission latency, which indicates no difference in time required for initiation of sexual activity. Additionally, no difference in mount frequency and intromission frequency was found. CONCLUSION: The present study provides scientific evidence for the traditional use of Lycium barbarum on male sexual behavior. The result provides basis for further study of wolfberry on sexual functioning and its use as an alternative treatment in reproductive medicine.postprintThe 30th Annual Meeting of the Australian Neuroscience Society, in conjunction with the 50th Anniversary Meeting of the Australian Physiological Society (ANS/AuPS 2010), Sydney, Australia, 31 January-3 February 2010. In Abstract Book of ANS/AuPS, 2010, p. 177, abstract no. POS-TUE-19

Combined effects of rearing environment and lead (Pb2+) exposure on visuospatial learning and memory in rats

Critical periods of neural development occur during early postnatal life that correspond with increases in synaptic plasticity and the formation of neural circuits needed for learning and memory. This development can be profoundly influenced by experience and negatively affected by environmental toxins. Environmental enrichment and lead exposure inversely affect mediators of synaptic plasticity, which suggests that enrichment may have an attenuating effect on lead induced cognitive deficits. A wealth of evidence has indicated that exposure to excessive amounts of inorganic Pb2+ during early development can produce long lasting cognitive deficits in humans. Evidence also suggests that children raised in an impoverished environment are at a disproportionate risk for developing Pb2+-induced cognitive deficits compared with peers exposed to an enriched environment. The present study evaluated the effects of both developmental Pb2+ exposure and environmental enrichment on visuospatial working and long-term memory in rats. Animals were fed either 1500 ppm Pb2+ acetate-laced rat chow or standard chow and exposed to either an impoverished environment (single housed, bedding only) or an enriched environment (4 rats/cage with toys, enclosures, etc.) for 7 weeks following weaning (PN day 25). Long-term and working memory error rates were assessed during a 17 day radial arm maze (RAM) learning task. Results suggest that the quality of the rearing environment but not Pb2+ exposure had a significant effect on learning performance. These findings suggest that the detrimental effects of Pb2+ exposure on cognitive development may be attenuated by exposure to an enriched environment and that the combination of being reared in an impoverished environment coupled with Pb2+ exposure can significantly impair learning performance later in life

Brain Derived Neurotrophic Factor Modulates Behavioral and Brain Responses to Social Stress

Social stress is a prevalent factor in society that can cause or exacerbate neuropsychiatric disorders including depression and posttraumatic stress disorder. According to the National Institutes of Health, 6.9% of adults in this country currently suffer from depression, and 4.1% suffer from an anxiety disorder. Unfortunately, current treatments are ineffective in reducing or alleviating symptoms in a majority of these patients. Thus, it is critical to understand how social stress changes in brain and behavior so that we might develop alternative treatments. Brain derived neurotrophic factor (BDNF), which binds to tyrosine kinase B (TrkB) receptors, plays a role in fear learning and in behavioral responses to stress, although we do not currently know whether BDNF promotes or prevents these responses. The purpose of this project was to understand how BDNF alters brain and behavior in response to social stress using a model of social stress in Syrian hamsters, termed conditioned defeat (CD). CD refers to the marked increase in submissive and defensive behavior following social defeat. Specific Aim (SA) 1 tested the hypothesis that BDNF, via TrkB receptors, promotes CD learning. Instead, we found that BDNF and a selective TrkB receptor agonist reduced CD and that a TrkB receptor antagonist enhanced CD. SA 2 tested the hypothesis that the behavioral response observed following systemic administration of TrkB-active drugs is mediated via their action in specific nodes of the neural circuit underlying CD. Unfortunately, the vehicle in which these drugs are dissolved independently activates immediate early gene expression making interpretation of these data impossible. Finally, SA 3 tested the hypothesis that BDNF alters defeat-induced neural activation at least in part by acting in the medial prefrontal cortex (mPFC). We demonstrated that BNDF microinjected into the mPFC site-specifically altered defeat-induced neural activation in the CD neural circuit supporting this hypothesis. Overall, these data suggest that BDNF acts to prevent social stress-induced changes in behavior, at least in part via the basolateral amygdala and the mPFC, and that BDNF-active drugs might be a useful avenue to pursue to discover new treatments for patients that suffer from stress-related neuropsychiatric disorders

Visual Pigments and Light Detection in the Eye

Most forms of animal vision begin with light absorption by visual pigments in the eye. A typical visual pigment consists of a G protein-coupled receptor protein – opsin – covalently conjugated to a chromophore. Sub-families of opsins show distinctive physicochemical properties and cellular expressions, often attuned to the specific visual functions that they serve. Here, we examined a number of molecular and functional features of three sub-families of opsins. We found that: (1) an active molecule of rhodopsin (a ciliary opsin expressed in rod photoreceptors for dim-light vision) amplifies the light signal by activating about 20-30 transducin molecules at the peak of the current response to single photon-absorption. (2) the thermal activation of native and some mutant rhodopsin and cone pigments (ciliary opsins in cone photoreceptors for color vision) in the dark is indeed an isomerization event, the rate of which can be quantitatively predicted by multi-vibrational-mode statistical mechanics. (3) melanopsin, a rhabdomeric opsin that underlies the intrinsic photosensitivity of a subgroup of retinal ganglion cells and is responsible for diverse non-image-forming visual functions in mammals, is also expressed in some thick, myelinated neuronal processes in the rat iris that possibly originate from the trigeminal ganglia. (4) neuropsin (OPN5), a previous orphan opsin, mediates the photoentrainment of the local circadian rhythm in the mammalian retina and cornea

Reversible silencing of spinal neurons unmasks a left-right coordination continuum.

This dissertation is focused on dissecting the functional role of two anatomically-defined pathways in the adult rat spinal cord. A TetOn dual virus system was used to selectively and reversibly induce enhanced tetanus neurotoxin expression in L2 neurons that project to L5 (L2-L5) or C6 (long ascending propriospinal neurons, LAPNs). Results focus on the changes observed during overground locomotion. The dissertation is divided into four chapters. Chapter One is a focused introduction to locomotion, including its broad description, the central mechanisms of its expression, how genetic-based approaches defined these mechanisms, and the limitations in these approaches. It concludes with details of the silencing paradigm used here and a summary of the main findings. Chapter Two describes the functional consequences of silencing L2-L5 interneurons. The focus is on selective disruption of hindlimb coordination during overground locomotion, revealing a continuum from walk to hop. These changes are independent of speed, step frequency, and other spatiotemporal features of gait. Left-right alternation was restored during swimming and stereotypic exploration, suggesting a task-specific role. Silencing L2-L5 interneurons partially uncoupled the hindlimbs, allowing spontaneous shifts in coordination on a step-by-step basis. It is proposed this pathway distributes temporal information for left-right hindlimb alternation, securing effective coordination in a context-dependent manner. Chapter Three focuses on the consequences of silencing LAPNs.Three patterns of interlimb coupling are disrupted: left-right forelimb, left-right hindlimb, and contralateral hindlimb-forelimb coordination. Observed again was a context-dependent continuum from walk-to-hop, irrespective of step frequency, speed, and the salient features that define locomotion. However, instead of spontaneous shifts in coordination as observed from L2-L5 interneuron silencing, the breadth of coupling patterns expressed were maintained on a step-by-step basis. It is proposed that this ascending, inter-enlargement pathway distributes temporal information required for left-right alternation at the shoulder and pelvic girdles in a context-dependent manner. Collectively, these data suggest that L2-L5 interneurons and LAPNs are key pathways that distribute left-right patterning information throughout the neuraxis. The functional role(s) of these pathways are exquisitely gated to the context at hand, suggesting that the locomotor circuitry undergoes functional reorganization thereby endowing or masking the silencing-induced disruptions to interlimb coordination

A cellular model for human daily behaviour

All the biochemical, physiological or behavioural processes whose period is about 24 hours possess a circadian rhythm. In mammals circadian rhythms control almost all aspects of human daily behaviour and physiology. Dysregulation of circadian rhythms leads to several pathologies, such as depression, cancer and metabolic syndromes. Mammalian circadian system is organized in a hierarchic fashion: suprachiasmatic nucleus (SCN) is master clock and governs the circadian rhythms of all peripheral oscillators, virtually all the other cells of the body. The study of human circadian rhythms in subjects in vivo is expensive, time consuming and invading. However, since SCN and peripheral oscillators share the same circadian molecular machinery, it is possible to use peripheral oscillator as model to study molecular mechanisms of circadian rhythms. To visualize in real-time cellular circadian rhythms, fibroblasts were infected with a lentivirus coding for the circadian reporter firefly luciferase under a clock gene promoter (Bmal1). After the synchronization of circadian rhythms, the measurement of the light emitted by the cells gave a representation of fibroblast circadian oscillations. The aim of the thesis was to establish the use of human primary skin fibroblasts as a valuable model to study different aspects of human circadian rhythms. To address these questions three projects were designed. A first set of experiments aimed at validating human skin fibroblast model, ascertaining that this in vitro model parallels in vivo human circadian parameters. We found a very good correlation between the in vivo and the in vitro period length in the three groups of subjects (two sighted and one blind) recruited for this study. Interestingly, although the in vivo period obtained from the blind group was longer than the in vivo period obtained from the sighted groups, the in vitro period length from the three groups of subjects was similar, revealing that human skin fibroblasts are insensitive to the after-effects caused by light. In summary, human circadian period can be approximated by measurement in fibroblasts. In a second project human age-related circadian impairments were studied in the cellular skin fibroblasts model. Indeed sleep-wake cycle alterations and phase advancing of gene expression and behaviour can be found in elder individuals. To better understand the rebound of ageing on the circadian rhythms we characterized the period length of skin fibroblasts from young and elder persons. No differences in amplitude, phase and period length were found between cells from the two groups. However, in the presence of sera from older donors human fibroblasts showed a reduced period length and a shorter phase of entrainment compared to the same cells measured in the presence of sera from young donors. These differences are likely due to one or more thermolabile substances, since heat-inactivation of sera from older donors almost undid the reduction of the circadian period length. Thus, these results suggest that during ageing the molecular machinery of peripheral circadian clocks does not change per se, but some age-related circadian changes observed in vivo might be caused by circulating molecules. Human fibroblasts were also used to investigate the role of melatonin as zeitgeber on peripheral oscillators. Melatonin is secreted in a circadian fashion and was demonstrated to regulate the SCN firing rate and to entrain the sleep-wake cycle of most mammals and humans. The circadian presence of melatonin is well conserved in all biological fluids, suggesting that melatonin may be one of the molecules that the master clock uses to synchronize peripheral oscillators. This hypothesis was tested in damped fibroblasts, using a wide range of concentrations of melatonin to restore the amplitude of the rhythms. However, no increase of amplitude or phase shift of the rhythms was observed after treating cells with melatonin. Moreover, the application of the hormone to newly synchronized oscillators decreased their bioluminescence. In summary, the experiments demonstrated that melatonin does not play a direct role as peripheral oscillator zeitgeber. In conclusion, the studies of the present thesis succeeded in revealing three primary findings: first, fibroblast circadian rhythms parallel human circadian physiology, such as circadian period length. Second, apparently, during ageing the molecular components of peripheral circadian clocks in skin fibroblasts do not change per se, but some age-related circadian changes observed in vivo might be caused by one or more heat-sensitive substances present in the blood of older subjects. Finally, melatonin does not possess direct synchronizing properties on peripheral oscillators like fibroblasts. In total, the present thesis revealed that primary human skin fibroblasts are an easily accessible, cheap and reliable model to enlighten our understanding of human circadian mechanisms

Using IEG's to uncover pathways for spatial learning in the rat.

This thesis concentrates on the induction of the immediate early genes zif268 and c-fos in a functional and dysfunctional brain network. Initial studies focused on the creation of a task to allow the study of immediate early gene activation after working memory. Previous studies using such a paradigm have compared the animals performing the experimental task with poorly matched control groups. Experiments in this thesis attempted to rectify this. A group of experiments using the water maze found decreases in Zif268 activation in the experimental group as compared to control groups, mainly in the hippocampus and some parahippocampal areas. This was believed to have arisen because of a streamlining of the brain network in the Working Memory group. An increase in c-Fos immunoreactivity was seen in the Working Memory group as compared to controls in prefrontal regions. Structural equation modelling analysis was performed, which allows immediate early gene counts to be used to analyse networks of brain regions. In the Working Memory group connections were seen between the parahippocampal regions and the subiculum that progressed via the hippocampus, indicating that the hippocampus was still engaged by the task. In the control group analysed no such hippocampal pathway was found. This water maze task was then used to study zif268-EGFP activation in a novel transgenic rodent model, where the Zif268 promoter drives the expression of a fluorescent protein EGFP. Activation of both EGFP and Zif268 immunoreactivity was seen in the CA1 region of the animals performing the control task. No EGFP activation was seen in this region in the Working Memory group even though EGFP expression was seen in other regions. The GFP protein was also able to be seen under direct visualisation in the CA1 and dentate gyrus region of control animals. Concerning the dysfunctional brain, gene expression was analysed in the retrosplenial cortex after NMDA lesions of the anterior thalamic nuclei. Previous research has shown that lesions produce a dramatic hypoactivity in the protein products of the immediate early genes c-fos and zif268. Microarray analysis of retrosplenial tissue revealed that as well as decreases in expression of genes related to repair and cell adhesion/neurogenesis, an increase in c-fos mRNA was seen in the lesion hemisphere of the brain. This pattern of expression is opposite to that of the protein. Possible reasons for this are discussed